Recent progress in determining the crystal structures of human cytochrome P-450 enzymes is rapidly making an understanding of active site dynamics and the oxidative mechanism the critical factors that limit our ability to predict substrate and inhibitor specificity. In a continuation of a long-standing program, we propose here to clarify the dynamic behavior of both bacterial and human cytochrome P-450 active sites. In general, bacterial enzymes are studied first to establish and validate new methodologies, or to exploit unique properties of the enzyme. The techniques are then to be applied to the study of CYP3A4, CYP4Aaa, and other human P-450 enzymes. The proposed work focuses on the use of new (and novel) technologies, including the site-specific incorporation of unnatural amino acids with NMR and fluorescent probes, the use of "Click" chemistry to modify P-450 proteins for spectroscopic analysis, and the use of nanodiscs to stabilize CYP4 enzymes for studies of the role of covalent heme binding. In parallel, we propose to continue our analysis of the features of the catalytic mechanism(s) of P-450 enzymes pertinent to the prediction of the regiospecificity of substrate oxidation by cytochrome P-450. The proposed work includes generation of the active oxidizing species by novel methods, replacement of the proximal iron ligand by a selenocysteine, and the further use of radical probes to clarify the identity and nature of the reactive oxygen species. As an adjunct to the mechanistic studies, we will characterize and utilize new extremophilic P-450 enzymes, as these proteins have proven to be particularly suitable for characterization of the oxidizing species. Cytochrome P-450 enzymes play key roles in the metabolism of drugs and xenobiotics, the synthesis of endogenous factors such as the sterol hormones and eicosanoids, and the inactivation of endogenous mediators such as retinoic acid. They are of major practical interest because they are a primary determinant of drug lifetime and action, are themselves important targets for therapeutic agents (e.g., fungal infections, cancer), are responsible for a broad range of drug toxicities, and have strong biotechnological potential. The ability to predict P-450 substrate and inhibitor specificity is central to all of these areas of P-450 involvement.